Mytilus galloprovincialis (Mollusca: Bivalvia) in a warm-temperate South African estuarine embayment

The distribution, population structure, condition and gonad indices, and the attachment strengths of the invasive Mediterranean mussel Mytilus galloprovincialis are described from the Knysna Estuary, South Africa. Mussels were found in the rocky intertidal at the mouth of the estuary and on all man-made hard substrata as far as 12?km from the mouth in 2013. However, mussel beds were found only in the lower reach of the estuary, where mussel densities were as high as 120 per 0.01?m². When compared to mussels from the embayment regions of the estuary, those at the estuary mouth, where wave action was high, were generally smaller, had significantly lower condition and gonad indices, and greater attachment strength. This suggests that mussels within the sheltered regions of the estuary invest more energy in growth and reproduction, and that this is one reason for their success as an invasive species in the Knysna estuarine embayment.     

An ultrastructural study of shell regeneration in Mytilus edulis (Mollusca: Bivalvia)

Female budgerigars, provided with nest-boxes and exposed to male vocalizations, lay more rapidly under a 14 L: 10 D photoregime than under regimes involving shorter light periods, though some birds will lay even when kept under continuous darkness. The photoperiod affects principally the interval between nest-box entry and egg-laying. Birds with previous breeding experience lay sooner than naive birds.     

Modulation of Mytilus trossulus (Bivalvia: Mollusca) larval survival and growth in culture. Short communication

Commercial importance and ability to live in a wide range of salinities have made the common mussel, Mytilus trossulus, a relevant model to study modulation of larval growth and development. We investigated the effects of various salinities combined with neomycin and ampicillin application on Mytilus larvae survival and growth. Both neomycin and ampicillin enhanced trochophore and veliger survival under condition of low salinity. The average veliger size was increasing in accordance with the increase of salinity. In case of neomycin treatment 3.6% of the larvae reached the pediveliger stage. No abnormalities of larval morphology of the FMRFamide and 5-HT systems occurred after 7 days of culturing with both antibiotics.     

Identification of Several Cytoplasmic HSP70 Genes from the Mediterranean Mussel (Mytilus galloprovincialis) and Their Long-Term Evolution in Mollusca and Metazoa

The HSP70 protein family consists one of the most conserved and important systems for cellular homeostasis under both stress and physiological conditions. The genes of this family are poorly studied in Mollusca, which is the second largest metazoan phylum. To study these genes in Mollusca, we have isolated and identified five HSP70 genes from Mytilus galloprovincialis (Mediterranean mussel) and investigated their short-term evolution within Mollusca and their long-term evolution within Metazoa. Both sequence and phylogenetic analyses suggested that the isolated genes belong to the cytoplasmic (CYT) group of the HSP70 genes. Two of these genes probably represent cognates, whereas the remaining probably represent heat-inducible genes. Phylogenetic analysis including several molluscan CYT HSP70s reveals that the cognate genes in two species have very similar sequences and form intraspecies phylogenetic clades, differently from most metazoan cognate genes studied thus far, implying either recent gene duplications or concerted evolution. The M. galloprovincialis heat-inducible genes show intraspecies phylogenetic clustering, which in combination with the higher amino acid than nucleotide identity suggests that both gene conversion and purifying selection should be responsible for their sequence homogenization. Phylogenetic analysis including several metazoan HSP70s suggests that at least two types of CYT genes were present in the common ancestor of vertebrates and invertebrates, the first giving birth to the heat-inducible genes of invertebrates, whereas the other to both the heat-inducible genes of vertebrates and the cognate genes of all metazoans. These analyses also suggest that inducible and cognate genes seem to undergo divergent evolution. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Yves Van de Peer] Elena Drosopoulou and Nikolas Nikolaidis contributed equally to the present report.     

Expression of thick filament proteins during ontogenesis of the mussel Mytilus trossulus (Mollusca: Bivalvia)

The appearance of thick filament proteins organized into supramolecular complexes was studied by SDS-PAGE and Western-blot analysis at different developmental stages of the mussel Mytilus trossulus. Paramyosin appeared at the egg stage, while twitchin and myorod appeared at the blastula stage (12 h after fertilization). In addition, RT-PCR analysis showed that the twitchin genes were expressed starting from the blastula stage. Thus, the proteins forming thick filaments of the contractile apparatus of mussel muscles are expressed long before the formation of the first well-organized muscle system of the veliger larvae (55 h). Further, the ratios actin/myosin heavy chain (MHC) and paramyosin/MHC at the veliger stage (96 h) distinctly differed from those in the adult mussel.     

Muscle and neuronal differentiation in primary cell culture of larval Mytilus trossulus (Mollusca: Bivalvia)

Molluscan in vitro technology allows the study of the differentiation of isolated cells undergoing experimental manipulations. We have used the immunofluorescence technique and laser scanning microscopy to investigate the organization of muscle proteins (actin, myosin, paramyosin, and twitchin) and the localization of neurotransmitters (serotonin and FMRFamide) in cultured mussel larval cells. Differentiation into muscle and neuron-like cells occurs during the cultivation of mussel cells from premyogenic and prenervous larval stages. Muscle proteins are colocalized in contractile cells through all stages of cultivation. The cultivation of mussel cells on various substrates and the application of integrin receptor blockers suggest that an integrin-dependent mechanism is involved in cell adhesion and differentiation. Dissociated mussel cells aggregate and become self-organized in culture. After 20 days of cultivation, they form colonies in which serotonin- and FMRFamide-immunoreactive cells are located centrally, whereas muscle cells form a contractile network at the periphery. The pattern of thick and thin filaments in cultivated mussel cells changes according to the scenario of muscle arrangement in vivo: initially, a striated pattern of muscle filaments forms but is then replaced by a smooth muscle pattern with a diffuse distribution of muscle proteins, typical of muscles of adult molluscs. Myogenesis in molluscs thus seems to be a highly dynamic and potentially variable process. Such a “flexible” developmental program can be regarded as a prerequisite for the evolution of the wide variety of striated and smooth muscles in larval and adult molluscs.     

Development of the larval muscle system in the mussel Mytilus trossulus (Mollusca,Bivalvia)

In the present study we examined muscle development throughout the complete larval cycle of the bivalve mollusc, Mytilus trossulus. An immunofluorescence technique and laser scanning confocal microscopy were used in order to study the organization of the muscle proteins (myosin, paramyosin, twitchin, and actin) and some neurotransmitters. The appearance of the muscle bundles lagged behind their nervous supply: the neuronal elements developed slightly earlier (by 2 h) than the muscle cells. The pioneer muscle cells forming a prototroch muscle ring were observed in a completed trochophore. We documented a well‐organized muscle system that consisted of the muscle ring transforming into three pairs of velar striated retractors in the early veliger. The striations were positive for all muscle proteins tested. Distribution of FMRFamide and serotonin (5‐HT) immunocytochemical staining relative to the muscle ring differed significantly: 5‐HT‐immunioreactive cells were situated in the center of the striated muscle ring, while Phe‐Met‐Arg‐Phe‐NH2 neuropeptide FMRFamid immunoreactive fibers were located in a distal part of this ring. Our data showed clearly that the muscle proteins and the neurotransmitters were co‐expressed in a coordinated fashion in a continuum during the early stages of the mussel development. Our study provides the first strong evidence that mussel larval metamorphosis is accompanied by a massive reorganization of striated muscles, followed by the development of smooth muscles capable of catch‐contraction.     

Absence of a complete block to polyspermy after fertilization of Mytilus galloprovincialis (Mollusca,Pelecypoda) oocytes

Mytilus galloprovincialis oocytes undergo monospermic fertilizations (1 sperm nucleus/oocyte) over a wide range of sperm-oocyte ratios beyond which the number of penetrating sperm increases either linearly or exponentially over 10 min. Artificial activation of oocytes by KCl or the ionophore A 23187, up to the polar body extrusion stage, allows successful fertilizations upon a subsequent insemination. No organized and complete detachment of supernumerary oocyte-bound sperm is detected after fertilization. Reducing the external Na⁺ concentration promotes a higher rate of fertilizations. These results suggest that no complete block to polyspermy is established in this species but that a partial block, Na⁺ dependent, might be sufficient to ensure monospermic fertilizations under natural conditions.     

Development of the muscle system and contractile activity in the mussel Mytilus trossulus (Mollusca, Bivalvia)

The development of contractile apparatus was subjected to comparative analysis during ontogenesis of the mussel Mytilus trossulus. Indirect immunofluorescence with the polyclonal antibody against mussel twitchin, a protein of thick filaments, and florescent phalloidin as a marker of filamentous cell actin were used to monitor changes in the developing muscle system at different larval stages. The first definitive muscle structures were found at the late trochophore stage (36 h after fertilization) and starting from the midveliger stage (96 h), striated muscles, which are never present in adult mussels, were distinctly seen. The striated muscle periodicity was 1.25 microm in both mussle larvae and adult scallop. The contractile activities of veliger and adult muscles were measured using an electronic signal-processing videosystem. This work is the first complex study of morphological, biochemical, and physiological characteristics of the muscle system in the larvae and adult mollusks.     

Plasmodium cynomolgi: the hsp 70 gene.

The hsp 70 gene of Plasmodium cynomolgi was isolated and characterized. As expected the gene is highly similar to that of the hsp 70 gene of Plasmodium falciparum (98% at the protein level, 82% at the nucleotide level). Surprisingly, the hsp 70 gene appears to be present in a single copy in all the P. cynomolgi strains tested, a finding that has implications for the parasite's ability to undergo a heat shock response.     

Isolation and characterization of a sea urchin hsp 70 gene segment

Three clones containing Paracentrotus lividus sea urchin DNA sequences which cross-hybridize to Drosophila heat shock protein (hsp) 70 gene were isolated. The sequence arrangements in the three cloned DNA inserts were compared by restriction and cross-hybridization analysis. The results showed that they contain four different genes related to one Drosophila hsp 70 gene. One of these genes was subcloned, and two of the isolated fragments were shown to hybridize to genomic DNA and to RNA from heat-treated sea urchin embryo.     

Use of monoclonal antibodies for identification of growth-controlling neuropeptides in the mussel Mytilus edulis (Mollusca: Bivalvia)

Monoclonal antibodies were developed against cerebral ganglia (CG) of the mussel Mytilus edulis by the immunization of mice with unpurified homogenates of these organs. The screening protocol of hybridoma was based upon immunohistological observations of cytocentrifugated ganglia cells. A panel of 29 monoclonal antibodies (MABs) specific of CG epitopes was harvested and subsequently used for the immunocytochemical study of CG cells. Several subpopulations of ganglia cells were specifically revealed by MABs. Identification of epitopes involved in growth control was approached via the application of a bioassay allowing the assessment of protein synthesis stimulation. MAB 42 and 46 affected amino acid incorporation induced by CG extract. These results lead to the conclusion that the epitopes recognised by these antibodies are involved in growth control. An immunoenzymatic assay was performed with CG extracts for quantitative analyses of epitopes.     

Reaction of the mussel Mytilus galloprovincialis (Bivalvia) to Eugymnanthea inquilina (Cnidaria) and Urastoma cyprinae (Turbellaria) concurrent infestation

In total 480 individuals of Mytilus galloprovincialis were sampled monthly from October 2009 to September 2010, at the shellfish farm in the Mali Ston Bay, south Adriatic Sea (Croatia) in order to assess the extent of pathology imposed by two parasites, Eugymnanthea inquilina (Cnidaria) and Urastoma cyprinae (Turbellaria). Although a deteriorating impact on host reproduction or condition index was lacking, we evidenced ultrastructural and functional alteration in host cells at the attachment site. Ultrastructural changes included hemocytic encapsulation of the turbellarian and cell desquamation in medusoid infestation. Caspase positive reaction inferred by immunohistochemistry (IHC) was triggered in cases of turbellarian infestation, in contrast with hydroids, suggesting that the former exhibits more complex host-parasite interaction, reflected in the persistent attempts of the parasite to survive bivalve reaction. We have evidenced that both organisms trigger specific host reaction that although not costly in terms of host reproductive cycle or growth, results in mild tissue destruction and hemocyte activation. A lower degree of tissue reaction was observed in cases of hydroid infestation, compared to turbellarian.     

Anthessius spp. (Anthessiidae) associated with Tridacnidae (Mollusca: Bivalvia) removed to a new genus Tridachnophilus

Genus Anthessius includes 36 species. Previous attempts to clarify the interspecific affinities reached no definite conclusions. Humes and Ho (1965) found that, based on the armature of the third exopod segment of the fourth leg, the known species could be arranged into two groups, but that this difference did not correspond with host preferences. The present study of A. alatus Humes and Stock shows that species of Anthessius associated with Tridacnidae differ from the rest (except A. fitchi Illg) in the marked indentation of the cephalosome and in the presence of well developed cephalosomal and metasomal epimeral lobes producing a very characteristic shape. They are here removed to a new genus, Tridachnophilus.     

Reconstructing the Anomalodesmata (Mollusca: Bivalvia): morphology and molecules

The extant anomalodesmatan bivalves have always proved rather enigmatic and difficult to interpret, both in terms of their relationships to other bivalve taxa and the interrelationships of the constituent families. These difficulties stem from their diverse and often highly specialized life habits which have resulted in a wide array of disparate morphologies, and also from the fact that many are extremely rare. Classifications based on morphological characters have been dogged by fears that convergent and parallel evolution has masked phylogenetic signals. Molecular surveys of members of 12 of the 15 constituent families, using the 18S rRNA gene, have revealed that anomalodesmatans are robustly monophyletic and lie within the basal heterodonts. The Anomalodesmata should no longer be regarded as a subclass, but as a part of the Heterodonta. Here we present an enhanced analysis of 32 anomalodesmatan species (representatives of 12 families). Our results, subjected to Maximum Parsimony, Maximum Likelihood and Bayesian analyses, challenge our understanding of the internal relationships within the Anomalodesmata. In particular they indicate the need for a re-distribution of the families traditionally placed in the Thracioidea and Pandoroidea into a 'thraciid' lineage (Thraciidae + Cleidothaeridae + Myochamidae) and a 'lyonsiid' lineage (polyphyletic Lyonsiidae + Clavagellidae + Laternulidae + Pandoridae). The endolithic Clavagella and endobenthic Brechites and Penicillus form a robust clade. The hypothesis that the carnivorous septibranchs are monophyletic can, thus far, be neither supported nor rejected. Mapping critical morphological characters onto our molecular results provides evidence of multiple loss of some characters (e.g. prismato-nacreous shell microstructure and shell spicules) and also multiple gain of others (e.g. chondrophores).  © 2006 The Linnean Society of London, Zoological Journal of the Linnean Society , 2006, 148 , 395–420.